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Discovery of a structural‑element specificG‑quadruplex “light‑up” probe

Zhao, Wei; Phan, Anh Tuân; Chang, Young‑Tae; Yoo, Jaeduk; Xu, Wang; Zhang, Liyun; Er,Jun Cheng; Ghosh, Krishna Kanta; Chung, Wan Jun

2014

Zhang, L., Er, J. C., Ghosh, K. K., Chung, W. J., Yoo, J., Xu, W., et al. (2014). Discovery of aStructural‑Element Specific G‑Quadruplex “Light‑Up” Probe. Scientific Reports, 4, 3776‑.

https://hdl.handle.net/10356/102230

https://doi.org/10.1038/srep03776

© 2014 Nature Publishing Group. This paper was published in Scientific Reports and ismade available as an electronic reprint (preprint) with permission of Nature PublishingGroup. The paper can be found at the following official DOI:[http://dx.doi.org/10.1038/srep03776]. One print or electronic copy may be made forpersonal use only. Systematic or multiple reproduction, distribution to multiple locationsvia electronic or other means, duplication of any material in this paper for a fee or forcommercial purposes, or modification of the content of the paper is prohibited and issubject to penalties under law.

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Discovery of a Structural-Element SpecificG-Quadruplex ‘‘Light-Up’’ ProbeLiyun Zhang1,2*, Jun Cheng Er1,3*, Krishna Kanta Ghosh1, Wan Jun Chung4, Jaeduk Yoo1, Wang Xu1,Wei Zhao5, Anh Tuan Phan4 & Young-Tae Chang1,6

1Department of Chemistry, National University of Singapore, 3 Science Drive 2 117543, Singapore, 2Hefei Institutes of PhysicalScience, Chinese Academy of Sciences, Hefei, Anhui 230031, P. R. China, 3Graduate School for Integrative Sciences andEngineering, National University of Singapore, Centre for Life Sciences, #05-01, 28 Medical Drive, 117456 Singapore, 4Divisionof Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore637371, Singapore, 5School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P. R. China,6Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), 138667, Singapore.

The development of a fluorescent probe capable of detecting and distinguishing the wide diversity ofG-quadruplex structures is particularly challenging. Herein, we report a novel BODIPY-based fluorescentsensor (GQR) that shows unprecedented selectivity to parallel-stranded G-quadruplexes with exposed endsand four medium grooves. Mechanistic studies suggest that GQR associates with G-quadruplex groovesclose to the end of the tetrad core, which may explain the dye’s specificity to only a subset of parallelstructures. This specific recognition favours the disaggregation of GQR in aqueous solutions therebyrecovering the inherent fluorescence of the dye. Due to its unique features, GQR represents a valuable toolfor basic biological research and the rapid discovery of novel, specific ligands that target similar structuralfeatures of G-quadruplexes.

Nucleic acid sequences rich in guanines (G) have a propensity to arrange into G-quadruplexes (G4)1. Thesenon-canonical structures have been implicated in transcription regulation and telomere maintenance;hence, they are targeted for cancer treatment and detection2,3. Conversely, engineered G4 oligonucleo-

tides can act as drug delivery vehicles4–6, materials for nanodevices7,8, support catalysts9 or even drugs for cancer,HIV and other diseases10. For these reasons, there is strong impetus to develop probes to improve our under-standing of G4 and their ligand binding characteristics11.

Studies suggest that G4 structures are highly polymorphic and exhibit distinct differences between eachother12,13. These myriad of topologies arises from the combination of several well-defined structural elementssuch as nucleic acid type (DNA or RNA), molecularity (monomer, dimer or tetramer), strand orientation(parallel, antiparallel or hybrid), loops (orientation, sequence and length), grooves type (narrow, wide and/ormedium), end-cap morphology and number of G-tetrads1,14. Efficient probing of these motifs provides oppor-tunities for discrimination and is also a prerequisite to the discovery and study of new G4 specific ligands15,16. Wewere thus motivated to develop fluorescent probes that are specific for these fundamental elements.

Several fluorescent probes capable of discerning G4 from duplex DNA have been developed. These emissiveprobes are generally planar structures which achieve selectivity through end-stacking with G-tetrads17–19. Incontrast, structural specific G4 probes are considerably rarer. Moreover, to achieve higher specificity in distin-guishing different G4s, simultaneous association with multiple structural elements is desired19. Reported probesare conceived from ligands which have been found to interact with the grooves of G4 a priori20–22. Therefore, suchrational design strategies can have limited efficiency for discovering new G4 probes with novel selectivity21.Conversely, employing the diversity-oriented fluorescent library approach (DOFLA) is a more effective strat-egy23–25; screening of diversity oriented fluorescent libraries (DOFL) can accelerate the identification of fluor-escent probes with the desired qualities without former insight on the structural guidelines needed.

ResultsSensor discovery. A collection of 5000 potential fluorescent sensors were thus gathered and an unbiased, highthroughput screening was performed to uncover primary hits responding to G4 (Fig. S1). Additionalmodifications to improve the quantum yield of the hit after binding led to the discovery of a sensitive andhighly selective fluorescent sensor for G4 (GQR, Fig. 1). In the presence of 93del – an interlocked, dimeric,

OPEN

SUBJECT AREAS:FLUORESCENT DYES

NUCLEIC ACIDS

Received25 November 2013

Accepted23 December 2013

Published20 January 2014

Correspondence andrequests for materials

should be addressed toY.-T.C. (chmcyt@nus.

edu.sg)

* These authorscontributed equally to

this work.

SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 1

parallel-stranded G4 with 4 medium grooves26 – GQR displayed upto 30-fold increase in fluorescence at 597 nm and a 12 nmbathochromic shift in emission maximum (Fig. 2). A Job plotanalysis of the GQR-93del complex revealed a bindingstoichiometry of 151 (Fig. S2); the dissociation constant (Kd) wasthus determined as 25.18 6 0.02 mM (Fig. 2, inset and Table S1).

Selectivity of GQR. To investigate the uniqueness of GQR, weexamined its selectivity towards various G4 oligonucleotides withdifferent structural elements (Table 1). Like 93del, J19, T95, T95and T95-2T form parallel-stranded G4 with exposed ends and fourmedium grooves (Fig. S3)26–29. When incubated with GQR, all foursequences elicited a fluorescence increase from the dye (Fig. 3).Specifically, the enhancement was most pronounced with 93del. Incontrast, both c-kit1 and Pu24T are parallel-stranded but theypossess additional snapback motifs that cap the G-tetrad core30,31.Interestingly, both sequences returned negligible fluorescence whenmixed with GQR (Fig. 3 and Fig. S4). On the contrary, Oxy and HTadopt non-parallel-stranded conformations (Fig. S3)32,33; GQRsimilarly remained quenched when incubated with either G4(Fig. 3 and Fig. S4). Likewise, GQR remained virtually non-fluorescent in the presence of other conventional nucleic acids(Fig. 3 and Fig. S4). Previous reports of parallel-stranded selectiveG4 sensors generally do not display selectivity for additional motifswithin the parallel-stranded structures20,22,34. To the best of ourknowledge, GQR is the first fluorescent dye that is able to discernamong various types of parallel-stranded G4.

Mechanism studies. Disaggregation-induced emission of GQR.Following experiments were aimed at understanding the sensingmechanism of GQR. First, we examined the photophysical

properties of GQR. GQR exhibited strong fluorescence emission inorganic solvents, but only negligible emission in buffer (Table 2,entries 13–16 vs. entry 1). Further measurements of the absorptionspectra showed significant peak broadening and a bathochromicshifts in buffer (Fig. 4a). With higher concentrations of GQR,more pronounced red-shifts were observed suggesting that thisphenomenon may be a result of dye aggregation (Fig. S5).Transmission electron microscope and dynamic light scatteringanalysis revealed the existence of GQR-aggregates with sizesrelated to the dye concentration (Fig. S6 and S7). Together, theseresults indicate that the low fluorescence of GQR in buffer is a resultof aggregation-caused quenching (ACQ)35 thereby affirming thepotential of GQR to behave as fluorescent turn-on sensors forG422,36,37. To confirm this potential, the absorption spectra of GQRwith 93del were studied. Indeed, when GQR was mixed withincreasing concentrations of the G4, the spectral shape graduallyshifted towards that similar to organic solvents (disaggregatedstate) (Fig. 4b).

Environmental sensitivity of GQR. To investigate the contribution ofenvironment polarity to the emission of GQR, we examined its emis-

Figure 1 | The structures of GQR and its analogues.Figure 2 | GQR reacts with 93del to give a turn-on fluorescence response.Fluorescence spectra of GQR (10 mM) upon incubation with serial

dilutions of 93del (from 0–80 mM) in buffer (20 mM K2HPO4/KH2PO4,

100 mM KCl, pH 7.0). lex: 360 nm. WF (without 93del) 5 0.014, WF (in

40 mM 93del) 5 0.28. Inset: photographic image of GQR (10 mM) in the

presence of 93del (from 0–40 mM) and data plot of fluorescent emission

intensity upon addition of 93del (from 0–80 mM). F0 and Fmax are the

fluorescent maximum intensities of the GQR in the absence and presence

of 93del respectively. Kd 5 25.18 6 0.02 mM (one site specific binding

model). Values are represented as means (n 5 3). Measurements were

taken at room temperature (RT).

Table 1 | Oligonucleotide sequences used in this work

Name Sequence (59 R 39) Structural Elements Origin

93del GGGG TGGG AGGA GGGT parallel, interlocked dimeric Aptamer, HIV-1 integraseJ19 GIGT GGGT GGGT GGGT parallel, dimeric Aptamer, HIV-1 integraseT95 GGGT GGGT GGGT GGGT parallel, dimeric Aptamer, HIV-1 integraseT95-2T TT GGGT GGGT GGGT GGGT parallel, monomeric Aptamer, HIV-1 integrasec-kit1 AGGG AGGG CGCT GGGA GGAG GG snap-back parallel c-kit oncogene promoterPu24T TGAG GGTG GTGA GGGT GGGG AAGG snap-back parallel c-myc oncogene promoterOxy GGGG TTTT GGGG antiparallel Oxytricha nova telomereHT TT GGGTTA GGGTTA GGGTTA GGGA hybrid Human telomeredsDNA Sigma-D8515 genomic dsDNA Calf thymusssDNA Sigma-D8899 genomic ssDNA Calf thymusRNA Sigma-R7250 single-stranded Calf liver

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SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 2

sion spectra in various toluene-methanol mixtures. We observed thatthe emission maximum of GQR red-shifted from 582 to 590 nm assolvent polarity decreases from methanol to toluene (Fig. S8). Theseobservations suggests that the bathochromic shift in the emission ofGQR when incubated with 93del is a consequence of the G4 provid-ing a hydrophobic binding pocket for GQR thereby affirming theinteraction between the two entities.

Binding mode studies. We were further interested in the interactionof GQR with its respective G4s at the molecular level. To this end,four additional analogues of GQR were studied in greater detail(Fig. 1). Like GQR, compounds 2, 3, 4 and 5 shared the same absor-bance spectra with GQR in DMSO (Fig. S9) and displayed highquantum yields (Table 2, entries 17–20). However, in buffer, only2 and 3 showed aggregation-induced absorption spectra (Fig. S9) andquenched quantum yields (Table 2, entries 21–24) analogous toGQR. Similarly, when incubated with 93del, only 2 and 3 similarlyexhibited a distinct enhancement in fluorescence albeit to a lesserextent than GQR (Table 2, entries 25–28 vs. 6). These results validatethe importance of disaggregation and the carboxamide group ofGQR in achieving effective fluorescence probing of G4.

Molecular docking was subsequently performed to understand thebinding mode38,39. As depicted in Fig. 5a, binding simulation suggeststhat GQR associates with the groove region of the 93del (PDB ID:1Y8D) core close to the ends. In contrast, a simulation of an end-stacking mode revealed a dramatically weaker affinity and higherenergy compared to a groove binding mode (Table S2). This resultis consistent with the general lack of peak shifts in the 1H iminoproton NMR spectra of 93del and T95-2T upon addition of GQR –an observation that contrasts that of end-stacking ligands (Fig.

S10)15,17,18. A closer examination of the binding model suggests plaus-ible hydrogen bonds with the carboxamide group which may accountfor the enhanced fluorescence of GQR compared to 2 and 3 (Fig. 5b).

DiscussionCommon BODIPY dyes have a tendency to aggregate which resultsin self-quenching40,41. This ACQ effect often limits the label-to-ana-lyte ratio and narrows the practical applications42. However,BODIPY can recover its high fluorescence in response to a targetmolecule through a recognition-induced disassembly of the aggre-gates. In this study, GQR-aggregates exhibit remarkable fluorescenceenhancement in the presence 93del. However, water-soluble analo-gues show no such response under the same conditions. The discov-ery of GQR as an effective fluorescent sensor demonstrates that theconventional ACQ problem can, in reality, be a general mechanismfor probe development43,44.

The structural polymorphism of G-quadruplexes has been sup-ported by NMR and X-ray crystallography. Nonetheless, a simplemethod to distinguish different G-quadruplex structures conveni-ently and sensitively is highly desirable. The use of specific fluor-escent probes, such as GQR, is one such strategy. Fluorescent probescan be potentially employed for mapping and tracking G-quadru-plexes-based structure motifs17,45 and can also be used for rapid dis-covery of novel, specific anticancer drugs that recognize similarG-quadruplex structural elements to the probe.

In summary, we have described the systematic discovery of a novelfluorescent dye – GQR – which specifically ‘‘light-up’’ when bound toparallel G4 with end exposed medium grooves. Specific recognitionand favourable binding to G4 induces the disaggregation of GQRwhich results in recovery of the inherent fluorescence of the dye.Preliminary mechanistic studies suggest a groove-binding modeclose to the end of the quadruplex core, thereby accounting for the

Figure 3 | Selectivity of GQR to oligonucleotides. (a) Fluorescence

titration of GQR with various G-quadruplexes, DNA and RNA.

Conditions: GQR (10 mM), nucleic acid (from 0 to 40 mM), buffer

(20 mM K2HPO4/KH2PO4, 100 mM KCl, pH 7.0), RT. lex: 360 nm, lem:

600 nm. (b) Photographic image of GQR (10 mM) mixed with various

nucleic acids (40 mM). Irradiation with a hand-held UV lamp at 365 nm.

Table 2 | Fluorescence quantum yields (WF) of respective probes invarious environments

Entry Probe Solvent Analytea WFb

1 GQR Bufferc - 0.0142 GQR Buffer 93del 0.283 GQR Buffer J19 0.224 GQR Buffer T95 0.195 GQR Buffer T95-2T 0.146 GQR Buffer c-kit1 0.0097 GQR Buffer Pu24T 0.0058 GQR Buffer Oxy 0.0279 GQR Buffer HT 0.00710 GQR Buffer dsDNA 0.01111 GQR Buffer ssDNA 0.00812 GQR Buffer RNA 0.01613 GQR DMSO - 0.5514 GQR MeOH - 0.5715 GQR PEG-400 - 0.4616 GQR Toluene - 0.3017 2 DMSO - 0.3018 3 DMSO - 0.3619 4 DMSO - 0.3920 5 DMSO - 0.2121 2 Buffer - 0.01322 3 Buffer - 0.03023 4 Buffer - 0.3324 5 Buffer - 0.2425 2 Buffer 93del 0.2026 3 Buffer 93del 0.2127 4 Buffer 93del 0.3428 5 Buffer 93del 0.23aAnalyte concentration: 40 mM.bValues are represented as means (n 5 3). Measurements were taken at room temperature (RT).cBuffer: 20 mM KH2PO4/K2HPO4, 100 mM KCl, pH 7.0.

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SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 3

high specificity. Therefore, GQR represents a promising tool for thefuture discovery of similar G4 groove binding ligands that can beimportant for disease studies, G4-based drug deliveries andnanotechnology.

MethodsProbe synthesis. Detailed description of the synthesis of each probe can be found inthe Supplementary Information. Each step was characterized by high-resolution massspectra, 1H and 13C NMR.

Nucleic acids. DNA oligonucleotides were synthesized and structurally verified byNMR spectroscopy as previously reported26–33. dsDNA, ssDNA and RNA werepurchased from Sigma Aldrich (product number D8515, D8899 and D7250respectively) and used as such. The nucleic acids were dissolved in buffer (20 mMK2HPO4/KH2PO4 100 mM KCl, pH 7.0). DNA concentration was expressed instrand molarity using a nearest-neighbor approximation for the absorptioncoefficients of the unfolded species.

Absorbance. UV/Vis absorption spectra of dyes and nucleic acids in buffer (20 mMK2HPO4/KH2PO4, 100 mM KCl, pH 7.0) were recorded from 200 to 700 nm using aSpectraMax M2 spectrophotometer.

Fluorescence. Fluorescence measurements were carried out on a SpectraMax M2spectrophotometer in 96-well plates by scanning the emission spectra between 540and 700 nm (lex 5 360 nm). All experiments were repeated three times. Dataanalysis was performed using Origin 8.0 (OriginLab Corporation, MA).

Quantum yield measurements. Quantum yields were calculated by measuring theintegrated emission area of the fluorescent spectra in its respective solvents andcomparing to the area measured for Coumarin 1 (reference) (10 mM), WF 5 0.73 in

ethanol (g 5 1.36), lex 5 360 nm. Quantum yields were calculated using theequation:

WsampleF ~W

referenceF

Fsample

Freference

� �gsample

greference

� �2Absreference

Abssample

� �

where F represents the area of fluorescent emission, g is the refractive index of thesolvent, and Abs is absorbance at the excitation wavelength. Emission was integratedbetween 540 and 700 nm.

Dissociation constant measurements. The Kd of GQR to the respective G-quadruplexes were analyzed by Origin 8.0 (OriginLab Corporation, MA) using thefollowing equation for a one site specific binding model:

y~ymaxxKdzx

Where y represents the fluorescence fold change of GQR, ymax the fluorescence foldchange of GQR when saturated with G-quadruplex and x the concentration of the G-quadruplex in mM.

Transmission electron microscope. GQR (10 mM) was first prepared in buffer anddeposited on a thin copper-support film, followed by drying in vacuo. Images of thesamples were obtained with JEOL JEM 3010 HRTEM microscope and operated at100 kV without any contrast agent.

Dynamic light scattering. The dynamic light scattering of different concentrationGQR and other compounds were measured at 25uC in buffer using quartz cell. Allmeasurements were performed in triplicate in Zetasizer Nano ZS.

Figure 4 | Absorption spectra of GQR. (a) GQR (10 mM) in various

organic solvents. (b) GQR (10 mM) upon incubation with serial dilutions

of 93del (from 0–40 mM) in buffer. Values are represented as means (n 5

3). Measurements were taken at RT.

Figure 5 | Molecular docking results of GQR binding to the 93del.Illustration of groove-binding mode of GQR with 93del residues. Carbon

atoms are colored in white for GQR, pink and green 93del; oxygen and

nitrogen atoms are colored in red and blue respectively. (b) Plausible

hydrogen bonds between GQR and 93del residues G10 and A12 (black

dashed lines). Carbon atoms are colored in white for GQR, pink and green

for 93del; oxygen and nitrogen atoms are colored in red and blue

respectively.

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SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 4

Nuclear magnetic resonance spectroscopy. Titration with GQR was performed on a600 MHz NMR Bruker spectrometers equipped with a cryoprobe at 25uC.Oligonucleotides (200 mM) were dissolved in buffer (20 mM K2HPO4/KH2PO4,70 mM KCl, D2O/H2O (159), pH 7.0). The spectra were recorded immediately aftereach addition of GQR. Water suppression was achieved using excitation sculpting.

Molecular modeling. The coordinates of 93del structures were retrieved from theProtein Data Bank (ID code 1Y8D). DNA structures were prepared for docking. TheGQR structure was optimized using the Gaussian03 program (B3LYP/6-31G* level).By using Autodock 4.0, docking studies were carried out with the Lamarckian geneticalgorithm following the procedure developed for G-quadruplex DNA and liganddocking38,39. Two rounds of simulation were performed. In the first round, simulatedannealing was used to find a rough binding mode of GQR with 50 runs while keepingall other parameters default. The search space was subsequently reduced and another200 runs were conducted to get a more precise result. Following the docking studies ofGQR with 93del quadruplex DNA, all the figures were rendered using PyMOL v0.99.

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AcknowledgmentsThis study was supported by intramural funding and grant 10/1/21/19/656 from theA*STAR (Agency for Science, Technology and Research, Singapore) Biomedical ResearchCouncil. L.Z. acknowledges the financial support from Natural Science Foundation ofChina (No. 11105150) and the Special Financial Grant from China Postdoctoral ScienceFoundation (No. 2013T60613). J.C.E. acknowledges the receipt of a NGS scholarship. Theauthors thank Dr. Animesh Samanta and Dr. Santanu Jana for their great help in TEManalysis and discussion.

Author contributionsY.T.C. and A.T.P. conceived the idea and directed the work. L.Z. and J.C.E. designed theexperiments and co-wrote the manuscript. L.Z. designed, screened and identified theG-quadruplex sensor and analyzed the photophysical properties and binding mechanism.J.C.E. and J.Y. designed, synthesized and characterized all dye compounds used in thisstudy. K.K.G. designed and synthesized the diversity-oriented fluorescent libraries. W.J.C.synthesized and characterized all G-quadruplex sequences. W.X. performed the DLSanalysis. W.Z. performed the molecular docking studies. All authors contributed to dataanalysis and manuscript writing.

Additional informationSupplementary information accompanies this paper at http://www.nature.com/scientificreports

Competing financial interests: The authors declare no competing financial interests.

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SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 5

How to cite this article: Zhang, L.Y. et al. Discovery of a Structural-Element SpecificG-Quadruplex ‘‘Light-Up’’ Probe. Sci. Rep. 4, 3776; DOI:10.1038/srep03776 (2014).

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SCIENTIFIC REPORTS | 4 : 3776 | DOI: 10.1038/srep03776 6